Calcitonins and related analogs, such as Elcatonin, are known polypeptides which can be employed for treating bone atrophy (see, e.g., U.S. Pat. No. 4,086,221). Naturally occurring calcitonins, such as eel, salmon or human calcitonin, are C-terminal amidated polypeptides which consist of 32 amino acids, the first and the seventh amino acids in each case being L-cysteines whose mercapto groups are connected to each other by the formation of a disulfide bridge. The natural calcitonins can be obtained, for example, by extraction from the mammalian thyroid gland (see, e.g., U.S. Pat. No. 5,428,129).
Elcatonin is a modified synthetic xe2x80x9ccarbaxe2x80x9d analog of calcitonin whose activity is comparable with that of eel calcitonin (Morikawa et al., Experienta, 32, 1004, (1976)). In contrast to eel calcitonin, Elcatonin lacks an amino terminal end and the disulfide bridge of eel calcitonin has been replaced by a xe2x80x94(CH2)5xe2x80x94 xe2x80x9ccarbon bridge.xe2x80x9d
Currently, a variety of processes are known for the preparation of Elcatonin using purely chemical methods. These chemical methods involve condensation of the corresponding amino acids or peptides (see, e.g., U.S. Pat. Nos. 4,086,221 and 5,428,129). The purely chemical methods, however, all suffer from the disadvantage that, due to the elaborate purification methods required, the Elcatonin is obtained in low yield and its preparation is consequently very expensive.
It would accordingly be beneficial to be able to avoid the disadvantages of the purely chemical methods in the preparation of Elcatonin through the use of a approach which includes the recombinant preparation of a portion of the molecule. This could be achieved, for example, if a simple process for the recombinant preparation of a C-terminal polypeptide fragment was available. The recombinantly synthesized C-terminal fragment could then be used as a starting peptide for the preparation of calcitonin or carba analogs such as Elcatonin. A partially recombinant strategy would also facilitate the synthesis of peptides/peptide analogs of calcitonin, Elcatonin and related analogs or derivatives which could potentially include non-natural amino acids.
The present invention is directed to a process for the recombinant preparation of a calcitonin fragment and the use of the fragment in the preparation of calcitonin and related analogs including carba analogs (referred to hereinafter as xe2x80x9ccalcitonin carba analogsxe2x80x9d), such as Elcatonin. The invention includes recombinantly forming a fusion protein which includes a target sequence linked to a carbonic anhydrase through a cleavage site. The target sequence includes a sequence of at least about 15 amino acids residues corresponding to a fragment from near the C-terminus of calcitonin or to a closely related analog of such a fragment. Typically, the target sequence includes an amino acid sequence corresponding to amino acids residues 10 through 32 of calcitonin or closely related analogs (collectively referred to hereinafter as a xe2x80x9c10-32 fragmentxe2x80x9d). The recombinantly formed fusion protein is subsequently cleaved with a cleavage reagent to produce a polypeptide including the target sequence. The cleavage reaction may be carried out by contacting the fusion protein with either a chemical cleavage reagent or an enzymatic cleavage reagent. The choice of a suitable cleavage reagent and the corresponding cleavage site incorporated into the fusion protein will depend on the particular target sequence and carbonic anhydrase sequence present in the fusion protein. Typically, the cleavage reagent and cleavage site are selected such that the amino acid sequence constituting the cleavage site does not appear in the amino acid sequence of either the target sequence or the carbonic anhydrase. For example, a cyanogen bromide cleavage at methionine would not be employed with a fusion protein which included the 10-32 fragment from porcine, bovine or sheep calcitonin.
The cleavage site is typically present in a linker sequence which connects the carbonic anhydrase and the target sequence. Alternatively, the fusion protein may include a construct in which the C-terminus of the carbonic anhydrase is connected directly to the N-terminus of the target sequence. This may occur where the C-terminal residue(s) of the carbonic anhydrase and the N-terminal residue(s) of the target sequence constitute a cleavage site which allows cleavage of the peptide bond between the two fragments. In addition to a cleavage site present in a linker sequence, the carbonic anhydrase portion of the fusion protein may also include a different cleavage site which permits the fusion protein to be cleaved to form a xe2x80x9cminifusion protein,xe2x80x9d i.e., a polypeptide having a C-terminal portion of the carbonic anhydrase still linked to the target sequence.
One embodiment of the invention includes a method for the recombinant preparation of polypeptides corresponding to amino acids 10-32 of calcitonin or related analogs (xe2x80x9c10-32 fragmentsxe2x80x9d). The method typically includes the recombinant preparation of a polypeptide fragment (xe2x80x9c10-32 fragment-Xxxxxe2x80x9d) of the formula:
A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-A23-A24-A25-A26-A27-Gly-A29-A30-A31-Pro-Xxx xe2x80x83xe2x80x83(SEQ ID NO:1) 
wherein A10 is Gly or Ser, A11 is Lys, Thr or Ala, A12 is Leu or Tyr, A13 is Ser, Thr or Trp, A14 is Gln, Lys or Arg, A15 is Glu, Asp or Asn, A16 is Leu or Phe, A17 is His or Asn, A18 is Lys or Asn, A19 is Leu, Tyr or Phe, A20 is Gln or His, A21 is Thr or Arg, A22 is Tyr or Phe, A23 is Pro or Ser, A24 is Arg, Gly or Gln, A25 is Thr or Met, A26 is Asp, Ala, Gly, or Asn, A27 is Val, Leu, Ile, Phe, or Thr, A29 is Ala, Val, Pro or Ser, A30 is Gly, Val or Glu, A31 is Thr, Val or Ala. The C-terminal -Xxx group is typically a C-terminal carboxylic acid (xe2x80x9cxe2x80x94OHxe2x80x9d), a C-terminal carboxamide (xe2x80x9cxe2x80x94NH2xe2x80x9d), or group capable of being converted into a C-terminal carboxamide, such as an amino acid residue or a polypeptide group (typically having from 2 to about 10 amino acid residues). The 10-32 fragment represented by residues A10 to A32 (SEQ ID NO:2) corresponds to residues 10 through 32 of the amino acid sequences for eel (SEQ ID NO:37), salmon I (SEQ ID NO:38), salmon II (SEQ ID NO:39), salmon III (SEQ ID NO:40), chicken (SEQ ID NO:41), human (SEQ ID No:42), rabbit (SEQ ID NO:43), porcine (SEQ ID NO:44), bovine (SEQ ID NO:45) and sheep (SEQ ID NO:46) calcitonin or closely related analogs (see the calcitonin sequences shown in FIG. 9). The present method may also be employed to recombinantly produce 10-32 fragments corresponding to modified calcitonin sequences. The modified calcitonin sequences may include one or more conservative amino acid substitutions in the natural amino acid sequence.
The 10-32 fragment may be utilized in the preparation of calcitonin and related analogs. The preparation typically includes the condensation of an N-terminal fragment of the formula: 
wherein A2 is Gly, Ser or Ala; A3 is Asn or Ser; A8 is Val or Met; R2 is xe2x80x94(CH2)4xe2x80x94 or xe2x80x94CH(NH2)CH2Sxe2x80x94Sxe2x80x94; and Y is OH, OR1, where xe2x80x94R1 is a lower alkyl group;
with a recombinantly-formed polypeptide of the formula:
A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-A23-A24-A25-A26-A27-Gly-A29-A30-A31-Pro-Xxx xe2x80x83xe2x80x83(SEQ ID NO:1)
wherein a A10 is Gly or Ser, A11 is wherein A10 is Gly or Ser, A11 is Lys, Thr or Ala, A12 is Leu or Tyr, A13 is Ser, Thr or Trp, A14 is Gln, Lys or Arg, A15 is Glu, Asp or Asn, Al16 is Leu or Phe, A17 is His or Asn, A18 is Lys or Asn, A19 is Leu, Tyr or Phe, A20 is Gln or His, A21 is Thr or Arg, A22 is Tyr or Phe, A23 is Pro or Ser, A24 is Arg, Gly or Gln, A25 is Thr or Met, A26 is Asp, Ala, Gly, or Asn, A27 is Val, Leu, Ile, Phe, or Thr, A29 is Ala, Val, Pro or Ser, A30 is Gly, Val or Glu, A31 is Thr, Val or Ala, and -Xxx is xe2x80x94OH, xe2x80x94NH2, an amino acid residue or a polypeptide group;
in the presence of a non-enzymatic coupling reagent to form a calcitonin-derivative having the formula: 
In one embodiment of the invention, the recombinantly-formed 10-32 fragment is condensed with a desaminononapeptide. The desaminononapeptide is a carba analog of an N-terminal calcitonin fragment and typically has the formula: 
wherein A2 is Gly, Ser or Ala; A3 is Asn or Ser; A8 is Val or Met; and Y is OH, OR1, where xe2x80x94R1 is a lower alkyl group (i.e., a C1-C6 alkyl group). The condensation reaction may be carried out using a chemical coupling reaction such as those described in U.S. Pat. Nos. 4,086,221 and 5,428,129, the disclosures of which are herein incorporated by reference. Chemical coupling agents are well known to those skilled in the art. Suitable chemical coupling agents include carbodiimides and a variety of other non-enzymatic reagents capable of reacting with the xcex1-carboxylic acid group of a peptide to form an activated carboxylic acid derivative and/or capable of catalyzing the condensation of an activated xcex1-carboxylic acid derivative with an N-terminal xcex1-amino group of another amino acid or polypeptide. Chemical coupling reactions in which the C-terminal xcex1-carboxylic acid of the desaminononapeptide has been converged to an acid azide, mixed acid anhydride, acid imidazole or active ester may be employed in the present invention. An especially effective method of coupling two peptide fragments is carried out in the presence of a carbodiimide and a reagent capable of forming an active ester, e.g., a mixture of dicyclohexylcarbodiimide (xe2x80x9cDCCxe2x80x9d) and either N-hydroxysuccinimide (xe2x80x9cHOSuxe2x80x9d) or 1-hydroxybenzotriazole (xe2x80x9cHOBtxe2x80x9d).
The recombinantly-formed 10-32 fragment employed in the condensation preferably has the formula:
A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-A23-A24-A25-A26-A27-Gly-A29-A30-A31-Pro-Xxx xe2x80x83xe2x80x83(SEQ ID NO:1)
wherein A10 through A31 and -Xxx are as defined herein.
The product of the coupling reaction is typically a calcitonin carba analog having the formula: 
wherein A10 through A31 and -Xxx are the same as defined herein for the desaminononapeptide (SEQ ID NO:3) or the 10-32 fragment-Xxx (SEQ ID NO:1). The coupling of the desaminononapeptide and the recombinantly-formed peptide is typically carried out in the presence of a non-enzymatic coupling reagent.
A preferred embodiment of the invention provides a method for the recombinant preparation and amidation of a polypeptide fragment (referred to herein as the xe2x80x9cECF2-amidexe2x80x9d) having the formula:
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 xe2x80x83xe2x80x83(SEQ ID NO:6)
and for coupling the ECF2-amide to an amino terminal fragment of Elcatonin (referred to hereafter as xe2x80x9cECF1xe2x80x9d), which has the formula: 
The present invention also provides a nucleic acid sequence which includes a sequence coding for amino acids 10-32 of calcitonin or a related analog. The nucleic acid sequence typically encodes a fusion protein which includes the 10-32 fragment linked to a carbonic anhydrase through a cleavage site. The portion of the gene encoding the 10-32 fragment is preferably designed using optimal codon usage for a targeted host cell, such as E. coli, S. cerevisiae or P. pastoris.